The overall goal of this project is to determine whether T lymphocytes in myasthenia gravis (MG) express a limited set of T cell receptor (TCR) molecules which could serve as the targets of specific immunotherapy. MG is characterized by muscle weakness caused by an immune response to self acetylcholine receptor (AchR). The immune response is initiated by AChR specific T lymphocytes and tissue damage is caused by specific antibodies. Current therapy consists of nonspecific immunosuppression which causes many side effects. Targeted immunosuppression of only those disease inducing T cells could be highly effective yet safe. Much has been learned about the T cell response in MG by studying TCR in a mouse model called EAMG. A vast number of individual TCR (> 109) are generated by the association of two polypeptide chains (alpha and beta) which are formed by rearrangement of gene elements called Valpha and Jalpha and Vbeta, Dbeta and Jbeta respectively. About 25 Vbeta genes exist in the mouse. We recently discovered that a single Vbeta gene is used by > 50% of the T cells involved in EAMG. The TCR protein product of this gene will be the target of immune therapy with a specific monoclonal antibody in a separate project. Such therapy should be effective in significantly reducing the immune response in EAMG while suppressing only about 4% (1/25) of the normal T cell population. We now propose to further study the T cell "repertoire" for AChR in EAMG and to extend our analysis to T cells involved in human MG. We will do this in four steps. (1) Vbeta6+ TCR genes involved in EAMG will be sequenced using a polymerase chain reaction (PCR) DNA amplification method to obtain information on the Vbeta-Dbeta- Jbeta junctional region presumed to make contact with the antigen, AChR. (2) the Vbeta genes used by the AChR specific T cells which are not Vbeta6+ will be identified, as will the Valpha genes associated with the Vbeta6+ and Vbeta6- TCR. The junctional regions of these molecules will also be sequenced. The result should be a testable model of how TCR structure and reactivity influence susceptibility to EAMG as well as providing other targets for specific therapy. Step (3) involves the identification of MG patients who might exhibit limited TCR expression as seen in EAMG. Our hypothesis is that only patients expressing the same HLA marker should be expected to show similar restricted TCR expression. MG patients will therefore be screened for an HLA marker, DQB*0201, which is known to be highly correlated with MG susceptibility. (4) AChR-specific T cell clones will be derived from MG patients with the specific HLA marker and analyzed for TCR Valpha and Vbeta usage. V gene identification will be assisted by a novel, rapid technique called multi-probe RNAse protection assay, which will be done by Dr. A. Theofilopoulos. Junctional sequences of human alpha and beta chains will then be determined after specific PCR amplification. The results from this project are likely to have a significant impact on the field of autoimmunity. A large amount of structure-function information on the TCR repertoire for a known autoantigen will be derived in mouse and man. Concepts of therapeutic approaches in autoimmunity based on limited TCR gene expression will be rigorously tested in a mouse model and, if found to be applicable, in a human population. These approaches may be applicable not only to MG but to diabetes, rheumatoid arthritis, multiple sclerosis and other illness thought to share common immunopathological features.